Summary: Lecture 1

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• Inside-out patch clamp

  Simply retracting a pipette that is in the cell-attached configuration causes a small vesicle of membrane to remain attached to the pipette. By exposing the tip of the pipette to air, the vesicle opens to yield a small patch of membrane with iets (former) intracellular surface exposed. This arrangement, called the inside-out patch recording configuration allows the measurement or single-channel currents with the added  benefit of making it possible to change the medium to which the intracellular surface of the membrane is exposed. 

• Outside-out patch clamp

  If the pipette is retracted while it is in the whole-cell configuration, a membrane patch is produces that has its extracellular surface exposed. This arrangement, called the outside-out recording configuration is optimal for studying how channel activity is influenced by extracellular chemica signals, such as neurotransmitter. 

• Two main possibilities of genetic manipulation

  • Foreign DNA exists in the cell in the form of a plasmid. More common in prokaryote hosts. Prokaryotes are unicellular organisms with a very simple cell structure, like bacteria and virusses.
  • Foreign DNA is intergrated into the host genome. It has to be part of the chains of DNA. Always the case in mammals. 

• Principle of recombinant technology (in bacteria)

  1. Isolation of the gene of interest
     
  2. Modification of the gene
     
  3. Insertion of the gene into a vector. A vector is another name for plasmid! We use enzymes to glue en cut plasmids.
  4. Insertion of the vector into cells of the organism to be modified > transformation
  5. Tests to isolate genetically modified organisms 

• Four classes of neurotransmitters

  1. Amino acids: glutamate and gaba are the main excitatory en inhbitatory neurotransmitters in the brain
     
  2. Amines
  3. Acetylcholine
  4. Neuro-peptides. Larger neurotransmitters. More moderatory effect. 

• Synthesis and inactivation of amino acids and amines

  Synthesis of eznymes in cell body, making of neurotransmitters is in the axon terminal. 
   
  Inactivation largely through local reuptake

• Synthesis and inactivation of neuro-peptides

  Inactivation through break-down and diffusion 

• Two types of  Amino Acid Transmitters 

  • Excitatory amino acids, like glutamate and aspartate
  • Inhibitory amino acisds, like GABA and glycine 

• Diffuse modulatory system: Serotonin

  Serotonergic (5-HT) cell bodies are relatively few and are concentrated in the raphe nuclei of the midbrain and the brainstem. Large areas of the brain are innervated by serotonergic fibers. Serotonin has been implicated in the control of sleep and wakefulness, mood, anxiety and many other functions.
  Drugs that inhibit the reuptake of serotonin, the so-called selective serotonin reuptake inhibitors or SSRIs, are often used to treat depression, anxiety and obsessive-compulsive disorder. 
  

  • Synthesis in raphe nuclei of the brain stem
  • Break down by monoamine oxidase (MAO) 
    

• Diffuse modulatory system: Dopamine

  Synthesis in substantia nigra (SN) and ventral tegmental area (VTA) of the midbrain. 
  
  Dopamine projections:
  

  • Nigro-striatal: Substantia nigra project to the dorsal stratium.
  • Meso-limbic: The VTA connects to more ventral striatum (n.accumbens), septum and amygdala
  • Meso-cortical: spread out over the frontal cortex more.

  
  
  Dopmaine functions
  

  • Nigro-striatal (SN -> dorsal striatum): Modulates cortical motor control and action selection.
  • Meso-limbic/-cortical (VTA -> ventral striatum [n.acc], septum, amygdala, prefrontal cortex): Reward processing, motivation, modulation of cognitive (executive) control